Sensors and Actuators B: Chemical
传感器和执行器 B:化学品

Volume 263, 15 June 2018, Pages 486-492
Sensors and Actuators B: Chemical

Real-time monitoring of microbial activity using hydrogel-hybridized carbon nanotube transistors

https://doi.org/10.1016/j.snb.2018.02.137Get rights and content 获取权限和内容

Highlights 突出

  • Microbial activity is monitored in real-time using hydrogel-hybridized carbon nanotube transistors.

  • The detected signal shows the microbial growth stages such as lag, log and stationary phases for fungi species.

  • The system allows the differentiation of fungi and yeast species according to their signal patterns.

  • Conductance of CNT-FETs change due to the metabolites and by-products from microbial activity.

Abstract 抽象

We demonstrate a unique system mainly composed of a carbon nanotube field-effect transistor (CNT-FET) integrated with malt extract agar (MEA) hydrogel for the real-time monitoring of microbial growth and activity. Consumption of nutrients and production of metabolites by microbial cells such as fungi or yeast results in the change of chemical properties of the hydrogel matrix, and this chemical change is detected by the underlying CNT-FET underneath the MEA hydrogel. In this study, we were able to distinguish the different growth phases (lag phase, log phase and stationary phase) of microbial organisms from the conductance measurement of the MEA-hybridized CNT-FET. Two fungal species (Aspergillus niger, Aspergillus versicolor), and one yeast species (Saccharomyces cerevisiae) were tested. The CNT-FET signals showed peculiar signal patterns according to the microbial species, enabling the classification of microbial species in terms of CNT-FET signal patterns. The CNT-FET signals were compared with pH change of the MEA hydrogel matrix, and the CNT-FET signal followed the microorganism activity more closely than the pH signal. We expect that this platform can serve as a suitable substitute of currently time-consuming, high-cost, and laborious microbial monitoring procedure and expedite the development of improved simultaneous monitoring of various fungal and/or bacterial organisms.

Keywords 关键字

Microbial activity
Real-time monitoring
Malt extract agar
Carbon nanotube


1. Introduction 1. 引言

Many researches have been performed in order to understand the metabolism of microorganisms during their growth, proliferation, death, materials consumed and produced during metabolism, gene information, and their response to diverse drugs and environmental conditions [[1], [2], [3], [4], [5]]. Furthermore, new ways of classifying microorganisms beyond their morphological classification is being investigated according to their nutrients and metabolites [[6], [7], [8], [9]]. In the metabonomic study of metabolites, where the effects of drugs or nutrients on the collective metabolite mass are investigated, techniques such as nuclear magnetic resonance (NMR), liquid chromatography-mass spectroscopy (LC–MS), gas chromatography–mass spectroscopy (GC–MS) and Fourier-transform infrared spectroscopy (FT-IR) are utilized [[10], [11], [12], [13], [14]]. However, these methods usually require time-consuming sample pre-treatment processes and long measurement time, making difficult to monitor the real-time activity level of the currently growing microorganisms on the culture media. Therefore, we need more effective tools and techniques for the real-time study of the growth of microorganisms and the effect of collective mass of metabolites. [3,11]. Meanwhile, there have been report on utilizing nanoscale materials to detect various chemicals. Recent studies of nanomaterials such as carbon nanotubes (CNTs) or graphene demonstrate the detection of biomolecules via selective receptors and sensitive electrical transducers [[15], [16], [17], [18], [19]]. Sensors based on these nanomaterials can detect microorganisms such as bacteria, fungus, virus or their metabolites such as aflatoxin or ochratoxin [[20], [21], [22], [23], [24], [25], [26]]. Most of these nanoscale sensors use specific receptors to impart high selectivity to specific target materials. However, few studies have been conducted of their utilization for the monitoring of total metabolite concentration by hybridization with hydrogel matrices [27,28].
为了了解微生物在生长、增殖、死亡过程中的代谢、代谢过程中消耗和产生的物质、基因信息以及它们对各种药物和环境条件的反应,已经进行了许多研究[[1]、[2]、[3]、[4]、[5]]。此外,正在研究根据微生物的营养成分和代谢物对微生物进行形态分类以外的新方法[[6],[7],[8],[9]]。在代谢物的代谢组学研究中,研究了药物或营养物质对集体代谢物质量的影响,使用了核磁共振(NMR)、液相色谱-质谱(LC-MS)、气相色谱-质谱(GC-MS)和傅里叶变换红外光谱(FT-IR)等技术[[10]、[11]、[12]、[13]、[14]]。然而,这些方法通常需要耗时的样品预处理过程和较长的测量时间,因此难以监测培养基上当前生长的微生物的实时活性水平。因此,我们需要更有效的工具和技术来实时研究微生物的生长和代谢物集体质量的影响。[3,11]. 同时,也有关于利用纳米级材料检测各种化学物质的报道。最近对碳纳米管(CNTs)或石墨烯等纳米材料的研究表明,通过选择性受体和灵敏的电换能器检测生物分子[[15],[16],[17],[18],[19]]。基于这些纳米材料的传感器可以检测微生物,如细菌、真菌、病毒或其代谢物,如黄曲霉毒素或赭曲霉毒素[[20]、[21]、[22]、[23]、[24]、[25]、[26]]。 这些纳米级传感器中的大多数使用特定的受体来赋予特定靶材高选择性。然而,很少有研究利用它们与水凝胶基质杂交来监测总代谢物浓度[27,28]。

In this study, we demonstrate a system composed of a hybrid structure of carbon nanotube field-effect transistor (CNT-FET) integrated with malt extract agar (MEA) hydrogel for the real-time monitoring of microbial growth and activity. Consumption of nutrients and production of metabolites by microbial cells such as fungi or yeasts change the chemical properties of the hydrogel matrix, and this change is detected by the underlying CNT-FET underneath the MEA hydrogel. As a result, we were able to distinguish the different growth phases (lag phase, log phase and stationary phase) of fungi and yeast from the conductance measurement of the MEA-hybridized CNT-FET. Two different fungal species, Aspergillus niger (A. niger), Aspergillus versicolor (A. versicolor), and one yeast species Saccharomyces cerevisiae (S. cerevisiae) were tested. The CNT-FET signals showed peculiar signal patterns according to the microbial species, presumably due to their different cell growing mechanisms. Our work aims in demonstrating the effect of total metabolites during microbial growth of known species. We utilize a hydrogel-CNT hybrid structure that measures the total effect of metabolites from the microbial organisms. This strategy enables the real-time monitoring of the microbial growth activity during cultivation in hydrogel, which is advantageous compared to conventional methods. The results were in accord with previous results carried out by conventional methods such as biomass dry weight measurement or direct microscopic counting [29,30]. Also, our system enabled in situ real-time monitoring of the microbial activity, which is advantageous compared to conventional methods. The CNT-FET signals were compared with the pH change of the MEA matrix, and the CNT-FET signal followed the microbial activity more closely than the pH signal. Since this strategy enables the monitoring of microbial activity in real time, we expect that this platform can serve as an aid or substitute of current time-consuming, high-cost, and laborious microbial monitoring systems.
在这项研究中,我们展示了一个由碳纳米管场效应晶体管(CNT-FET)与麦芽提取物琼脂(MEA)水凝胶集成的混合结构组成的系统,用于实时监测微生物的生长和活性。微生物细胞(如真菌或酵母)对营养物质的消耗和代谢物的产生会改变水凝胶基质的化学性质,并且这种变化由MEA水凝胶下方的底层CNT-FET检测到。因此,我们能够从MEA杂交CNT-FET的电导测量中区分真菌和酵母的不同生长阶段(滞后期、对数期和固定期)。测试了两种不同的真菌物种,黑曲霉 (A. niger)、花色曲霉 (A. versicolor) 和一种酵母菌酿酒酵母 (S. cerevisiae)。根据微生物种类的不同,CNT-FET信号显示出特殊的信号模式,这可能是由于它们不同的细胞生长机制。我们的工作旨在证明总代谢物在已知物种微生物生长过程中的影响。我们利用水凝胶-CNT混合结构来测量微生物中代谢物的总效应。该策略可以实时监测水凝胶培养过程中的微生物生长活性,与传统方法相比具有优势。结果与先前通过生物量干重测量或直接显微计数等常规方法进行的结果一致[29,30]。此外,我们的系统能够对微生物活性进行原位实时监测,与传统方法相比,这是有利的。 将CNT-FET信号与MEA基质的pH变化进行对比,CNT-FET信号比pH信号更接近微生物活性。由于该策略能够实时监测微生物活动,我们希望该平台可以作为当前耗时、高成本和费力的微生物监测系统的辅助或替代品。

2. Materials and methods 2.材料与方法

2.1. Fabrication of MEA-hybridized CNT-FET devices and electrical characterization
2.1. MEA杂化CNT-FET器件的制造和电气特性

The CNT-FETs were fabricated in wafer-scale and each CNT channel had dimensions of 10 μm length and 3 μm width using a previously reported process (Fig. S1a) [31]. In brief, a nonpolar molecular pattern of octadecyltrichlorosilane (OTS) was formed on SiO2 (thickness 100 nm) solid substrate. The substrate was dipped in 0.05 mg/mL single-walled CNT (Hanwha Chemical Co., Ltd., Korea) solution in dichlorobenzene. The CNTs were assembled on the SiO2 regions, avoiding the nonpolar OTS regions. Finally, Ti/Au (10/30 nm) electrodes were deposited via thermal deposition and metal lift-off process. The MEA film was prepared by dissolving MEA (BD Difco, USA) in deionized water at 33.8 g/L concentration followed by autoclaving. The MEA-hybridized CNT-FETs were prepared by positioning the samples in a Teflon™ custom-made device holder and pouring 2 mL of the autoclaved MEA on the MEA well (Fig. S2). The MEA was cooled down to form a hydrogel on the CNT-FET with thickness of 20 mm. When needed, a separate pH probe (L6880, SI Analytics, USA) was imbedded in the MEA matrix for the simultaneous monitoring of CNT- FET and pH signal. A probe station (MST-4000a, MS-TECH) equipped with a semiconductor parameter analyzer (Keithley 4200) was utilized to measure the basic electrical characteristics such as I–V curve, gate property, and on-off ratio of the fabricated CNT-FET devices before and after MEA hybridization.
CNT-FET以晶圆级制造,每个CNT通道的尺寸为10μm长和3μm宽,使用先前报道的工艺(图S1a)[31]。简而言之,在SiO 2 (厚度100 nm)固体衬底上形成了十八烷基三氯硅烷(OTS)的非极性分子图谱。将底物浸入0.05 mg/mL单壁CNT(韩华化学株式会社,韩国)的二氯苯溶液中。碳纳米管组装在SiO 2 区域,避开了非极性OTS区域。最后,通过热沉积和金属剥离工艺沉积了Ti/Au(10/30 nm)电极。MEA薄膜的制备方法是将MEA(BD Difco,USA)溶解在浓度为33.8 g/L的去离子水中,然后进行高压灭菌。通过将样品放置在特氟龙™定制设备支架中并将 2 mL 高压灭菌的 MEA 倒入 MEA 孔中来制备 MEA 杂化 CNT-FET(图 S2)。将MEA冷却,在CNT-FET上形成厚度为20 mm的水凝胶。必要时,将单独的pH探头(L6880,SI Analytics,USA)嵌入MEA矩阵中,以同时监测CNT-FET和pH信号。利用配备半导体参数分析仪(Keithley 4200)的探针站(MST-4000a,MS-TECH)测量了MEA杂化前后制备的CNT-FET器件的基本电气特性,如I-V曲线、栅极特性和开关比。

2.2. Experimental setting of real-time microbial activity monitoring system
2.2. 实时微生物活性监测系统的实验设置

The CNT-FET was mounted on the device holder. Then, MEA film was formed on top of the CNT channel, as described above. After gelation, 1 mg/mL fungal solution was applied above the CNT channel region of the MEA-hybridized CNT-FET. We dropped 20 μL of 1 mg/mL A. niger solution on the MEA just above the CNT channel region of the MEA-hybridized CNT-FET. The device holder with CNT-FET was placed in an incubator and kept at constant temperature of 25 °C. The growth of the applied fungi was monitored both electronically and optically. The current of the CNT-FET and MEA pH change was monitored in real time using a two-channel source measure unit (Keithley 2636A). The current signal precision was 100 fA. Each channel was assigned to the CNT-FET and pH probe, respectively. A Labview (National Instruments, USA) program was used to monitor and record the real-time signals from the two channels. The morphological change of the fungal colony was monitored with a web camera (Plugable Tech., USA). The A. niger and A. versicolor solution used in this research were prepared by culturing the spores at 25 °C for 6 days on MEA. The Fungi were harvested with sterile loops and washed twice in pH 7.2 PBS buffer. Total fungi mass was determined with a ultramicrobalance (XF2U, Mettler Toledo, USA) [25]. The yeast S. cerevisiae was purchased from Sigma and dissolved in PBS without further purification.
CNT-FET安装在器件支架上。然后,如上所述,在CNT通道的顶部形成MEA薄膜。凝胶化后,将 1 mg/mL 真菌溶液施加在 MEA 杂交 CNT-FET 的 CNT 通道区域上方。我们将 20 μL 的 1 mg/mL 黑曲霉溶液滴在 MEA 上,就在 MEA 杂交 CNT-FET 的 CNT 通道区域上方。将带有CNT-FET的设备支架置于培养箱中,并保持在25°C的恒温下。 通过电子和光学方式监测应用真菌的生长。使用双通道源测量单元 (Keithley 2636A) 实时监测 CNT-FET 和 MEA pH 值变化的电流。电流信号精度为100 fA。每个通道分别分配给CNT-FET和pH探头。Labview(美国国家仪器公司)程序用于监控和记录来自两个通道的实时信号。用网络摄像头(Plugable Tech.,USA)监测真菌菌落的形态变化。本研究中使用的黑曲霉和花色曲霉溶液是通过在 25 °C 下在 MEA 上培养孢子 6 天来制备的。用无菌环收获真菌,并在pH 7.2 PBS缓冲液中洗涤两次。用超微天平(XF2U,Mettler Toledo,USA)测定真菌总质量[25]。酵母酿酒酵母购自Sigma,溶解在PBS中,无需进一步纯化。

2.3. MEA composition analysis
2.3. MEA组成分析

The change in composition of the MEA matrix was monitored using FT-IR and UV/Vis spectroscopy. The change in absorbance was monitored using a UV/Vis spectrophotometer (Cary 8454, Agilent, Inc.) in the wavelength range of 200 nm to 1100 nm. The FT-IR analysis on the effect of citric acid was carried out using FT-IR spectrophotometer (LabRam Aramis IR2, Horiba, Inc.).
使用傅里叶变换红外光谱和紫外/可见光谱监测MEA基质的组成变化。使用紫外/可见分光光度计(Cary 8454,Agilent, Inc.)在200nm至1100nm的波长范围内监测吸光度的变化。使用FT-IR分光光度计(LabRam Aramis IR2,Horiba,Inc.)对柠檬酸影响进行FT-IR分析。

2.4. CNT-FET response and pH change experiment
2.4. CNT-FET响应及pH变化实验

For the CNT-FET response and pH change when exposed to citric acid and ethanol, we first prepared a MEA-hybridized CNT-FET. Then, we sequentially dropped 5 μL of 0.1 M citric acid or 0.1 M ethanol in 20 s intervals.
对于暴露于柠檬酸和乙醇时的CNT-FET响应和pH变化,我们首先制备了MEA杂化CNT-FET。然后,我们以 20 秒的间隔依次滴入 5 μL 0.1 M 柠檬酸或 0.1 M 乙醇。

3. Results and discussion
3. 结果与讨论

Using the MEA-hybridized CNT-FET, we monitored the microbial activity of A. niger (Fig. 1). The AFM topography image of the SWNT channel having a width of 3 μm proves that a well-oriented SWNT network channel was formed between source and drain (Fig. S1b). From the SEM image, we observed that the SWNT channel is well formed on the silicon oxide layer and well coupled with the MEA hydrogel (Fig. S3). The current-voltage (IV) curves showed current levels of a few μA at 1 V DC bias (Fig. S4). The resistance of 81 CNT-FET devices showed a log-normal distribution with average values of ∼6 MΩ, which is a peculiar property of a percolating conductive network [35,36].
使用MEA杂交的CNT-FET,我们监测了黑曲霉的微生物活性(图1)。宽度为3 μm的SWNT通道的AFM形貌图像证明,在源极和漏极之间形成了一个方向良好的SWNT网络通道(图S1b)。从SEM图像中,我们观察到SWNT通道在氧化硅层上形成良好,并与MEA水凝胶很好地耦合(图S3)。电流-电压 (IV) 曲线显示,在 1 V DC 偏置下,电流水平为几 μA(图 S4)。81个CNT-FET器件的电阻呈现对数正态分布,平均值为∼6MΩ,这是渗流导电网络的特殊特性[35\u201236]。

Fig. 1
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Fig. 1. Conceptual schematic of microbial activity monitoring system using carbon nanotube field-effect transistors (CNT-FETs) hybridized with malt extract agar (MEA). The current and pH signal change was simultaneously monitored using a two channel source measure unit connected to a laptop computer running a computerized program.
图 1.使用碳纳米管场效应晶体管 (CNT-FET) 与麦芽提取物琼脂 (MEA) 杂交的微生物活性监测系统的概念示意图。使用连接到运行计算机程序的笔记本电脑的双通道源测量单元同时监测电流和 pH 信号的变化。

The A. niger site showed no significant growth up to one day. Then, it showed a rapid growth after one day of latency, as confirmed by optical images (Supporting Movie 1). This was further confirmed by observing the reflective light intensity of an A. niger colony on a culture cell. The light intensity dropped after 24 h (Fig. S5). It is reported that Aspergillus species have about 1 day of lag phase, followed by spore-generating log phase, and finally a stationary phase, where fungal growth and death occur simultaneously [32]. The current measured with our MEA-hybridized CNT-FET clearly shows a signal pattern in accordance to this phase development. As shown in Fig. 2a, the current shows no significant change for about 1 day, and afterwards, it shows a rapid current increase and maintains a saturated value for up to 3 days. When the pH was monitored simultaneously, the pH showed relatively rapid decrease to a more acidic condition and then maintained constant, which is in contrast to the CNT-FET signal change. (Fig. 2b) The pH change in case of A. niger can be explained by noting that citric acid is one of the main metabolites generated during the growth of A. niger and therefore the pH of the growth media decreases [33,34]. However, since citric acid is not the only metabolite of A. niger, the signal change of our CNT-FET will be due to the overall effect from all the metabolites and not only from the citric acid. In the absence of A. niger, both CNT-FET signal and pH did not change significantly (Fig. S6). This means that the signal change observed in Fig. 2a is due to microbial activity and not from any degradation effect of our system. To check the feasibility of using our sensors, we performed repeatability (Fig. S7) and reproducibility test (Fig. S8). To show the repeatability, yeast (20 μL, 1 mg/mL concentration) was applied to a fresh MEA-CNT channel region. After 24 h, the hydrogel was removed. Then, a fresh MEA gel was reapplied and the experiment was repeated. The current signal was monitored for an additional 12 h. We observed rapid growth from the first 8 h after culturing the yeast and we observed rapid growth again when washed and re-cultured, which confirmed that our method had sufficient repeatability. To show the reproducibility, we performed the experiments on A. niger for several samples. We prepared three different MEA-hybridized CNT-FETs. Microbial activity from the three devices showed similar patterns (Fig. S8).
A. niger 站点在一天内没有显着生长。然后,它在一天的延迟后显示出快速增长,正如光学图像所证实的那样(支持电影 1)。通过观察培养细胞上黑曲霉菌落的反射光强度,进一步证实了这一点。24小时后光强度下降(图S5)。据报道,曲霉属有大约1天的滞后期,然后是孢子产生对数期,最后是固定期,真菌生长和死亡同时发生[32]。使用我们的 MEA 混合 CNT-FET 测量的电流清楚地显示了符合该阶段发展的信号模式。如图2a所示,电流在大约1天内没有明显变化,之后,电流快速增加,并保持饱和值长达3天。当同时监测pH值时,pH值显示出相对快速的下降到更酸性的状态,然后保持恒定,这与CNT-FET信号的变化相反。(图 2b)黑曲霉的pH值变化可以通过注意到柠檬酸是黑曲霉生长过程中产生的主要代谢物之一来解释,因此生长培养基的pH值降低[33,34]。然而,由于柠檬酸不是黑曲霉的唯一代谢物,我们的CNT-FET的信号变化将是由于所有代谢物的整体影响,而不仅仅是柠檬酸。在没有黑曲霉的情况下,CNT-FET信号和pH值均无显著变化(图S6)。这意味着在图2a中观察到的信号变化是由于微生物活动,而不是来自我们系统的任何降解效应。为了检查使用传感器的可行性,我们进行了重复性测试(图S7)和再现性测试(图S8)。 为了显示重复性,将酵母(20 μL,1 mg/mL浓度)施加到新鲜的MEA-CNT通道区域。24小时后,除去水凝胶。然后,重新涂抹新鲜的MEA凝胶并重复实验。对当前信号进行了额外的12小时监测。我们从培养酵母后的前8小时开始观察到快速生长,并且在洗涤和重新培养时再次观察到快速生长,这证实了我们的方法具有足够的可重复性。为了证明可重复性,我们对几个样品进行了对黑曲霉的实验。我们制备了三种不同的MEA杂化CNT-FET。来自这三种装置的微生物活性显示出相似的模式(图S8)。

Fig. 2
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Fig. 2. Result of fungal growth activity using MEA-hybridized CNT-FETs and the pH change of the MEA hydrogel matrix for two different fungal species: Aspergillus niger (A. niger) and Aspergillus versicolor (A. versicolor). (a) The CNT-FET current and (b) pH change of the MEA matrix in time for A. niger. (c) The CNT-FET current and (d) MEA pH change in time for A. versicolor. The left and right inset in (a) and the inset in (c) shows the optical micrograph of an MEA-hybridized CNT-FET with A. niger after 24 h, 72 h and A. versicolor after 72 h growth, respectively (scale bar: 3 mm). The dashed lines in the inset in (a) and (c) shows the position of the electrodes for optical guidance.
图 2.使用 MEA 杂交 CNT-FET 的真菌生长活性结果以及两种不同真菌物种的 MEA 水凝胶基质的 pH 值变化:黑曲霉 (A. niger) 和花斑曲霉 (A. versicolor)。(a) 黑曲霉 MEA 基质的 CNT-FET 电流和 (b) pH 值变化。(c) CNT-FET电流和(d)MEA pH值随时间的变化。(a)中的左侧和右侧插图以及(c)中的插图分别显示了MEA杂交的CNT-FET与黑曲霉生长24小时、72小时和72小时后花斑曲霉的光学显微照片(比例尺:3 mm)。(a)和(c)中插图中的虚线表示用于光学引导的电极的位置。

We observed the species-selectivity of MEA-hybridized CNT-FET by observing the signal change under the growth of another fungal species, A. versicolor. The fungal species A. versicolor is reported to have a lag phase of about one day but a slower growth rate compared to A. niger [35]. As shown in Fig. 2c, the A. versicolor signal also showed the three-phase regions with a slower current change rate in the log and stationary phase, which is in accordance with the reported growth property of A. versicolor [36]. In contrast, the pH showed relatively fast change to a more acidic state at the beginning of the log-phase, and then it was followed by a plateau (Fig. 2d). These results show that the pH information alone is not sufficient in distinguishing the phase change and the effect of total metabolites during the growth of fungal species. Also, it shows that our MEA-hybridized CNT-FETs show signal patterns that reflect the growth phases reported in the literature [35].
我们通过观察另一种真菌物种 A. versicolor 生长下的信号变化来观察 MEA 杂交 CNT-FET 的物种选择性。据报道,真菌种类A. versicolor的滞后期约为1天,但与黑曲霉相比,其生长速度较慢[35]。如图2c所示,花斑曲霉信号也显示了对数和固定相中电流变化率较慢的三相区域,这与报道的花花曲霉的生长特性一致[36]。相比之下,pH值在对数阶段开始时显示出相对较快的酸性状态变化,然后是平台期(图2d)。这些结果表明,仅靠pH信息不足以区分真菌生长过程中的相变和总代谢物的影响。此外,它还表明,我们的MEA杂交CNT-FET显示出反映文献中报道的生长阶段的信号模式[35]。

To determine the effect of the MEA-implementation and fungal growth on sensor signals, the electrical properties of the CNT-FETs were observed before and after MEA-implementation and after the growth of A. niger on the MEA for 3 days. The MEA-implementation process had no adverse effects on the electrical properties of the CNT-FET devices (Fig. S9). The IV characteristics showed an increase of nonlinearity in the IV curves (Fig. S9a) and gate characteristics showed an increase of conductivity in the drain-source voltage to 0.1 V with the application of MEA. This is probably due to increase in the Schottky barrier between CNT/metal interface [37]. In both cases, the devices showed no significant change in gate characteristics (Fig. S9b) due to fungal growth.
为了确定MEA实施和真菌生长对传感器信号的影响,在MEA实施前后以及黑曲霉在MEA上生长3 d后观察CNT-FET的电学特性。MEA实施过程对CNT-FET器件的电气性能没有不利影响(图S9)。IV特性显示IV曲线中的非线性增加(图S9a),栅极特性显示,随着MEA的应用,漏源电压的电导率增加到0.1 V。这可能是由于CNT/金属界面之间的肖特基势垒增加[37]。在这两种情况下,由于真菌生长,器件的栅极特性没有显着变化(图S9b)。

To elucidate the reaction mechanism of the MEA-hybridized CNT-FET device, we performed spectroscopic analysis on the growth agar medium (Fig. 3). First, we measured UV/Vis spectroscopy of agar medium before and after A. niger growth (Fig. 3a). As a result, we could identify a peak at 230 nm that can be attributed to citric acid. It is reported that, in case of A. niger, citric acid is generated as a major metabolite [38]. Indeed, the UV/Vis spectrum of citric acid shows a peak at ∼230 nm wavelength when tested in water (Fig. 3b). The reduced peak around 270 nm can be attributed to the consumed materials in the MEA. The production of citric acid in the MEA by A. niger was also confirmed by FT-IR analysis. The FT-IR spectrum showed three peaks corresponding to Csingle bondO stretch (1174 cm−1), Csingle bondH rock (1365 cm−1), Cdouble bondO stretch (1750 cm−1) of citric acid [39].
为了阐明MEA杂化CNT-FET装置的反应机理,我们对生长琼脂培养基进行了光谱分析(图3)。首先,我们测量了黑曲霉生长前后琼脂培养基的紫外/可见分光光谱(图3a)。因此,我们可以在230nm处确定可归因于柠檬酸的峰。据报道,在黑曲霉的情况下,柠檬酸是主要代谢产物[38]。事实上,在水中测试时,柠檬酸的紫外/可见光谱在 ∼230 nm 波长处显示出峰值(图 3b)。270 nm 附近峰的降低可归因于 MEA 中消耗的材料。傅里叶变换红外分析也证实了黑曲霉在MEA中产生的柠檬酸。傅里叶变换红外光谱显示,柠檬酸的C single bond O拉伸(1174 cm −1 )、C single bond H岩石(1365 cm −1 )、C double bond O拉伸(1750 cm −1 )对应3个峰[39]。

Fig. 3
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Fig. 3. Spectroscopy analysis on MEA before and after A. niger growth. (a) UV/Vis absorbance spectrum of MEA before (black) and after (red) growth, and difference of the two spectrum (blue). (b) UV/Vis absorbance spectrum of citric acid in water for different concentrations. (c) FT-IR spectrum of sampled MAE before (black) and after (red) A. niger growth. The peaks at 1174, 1365, 1750 cm−1 show typical peaks for citric acid. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
图 3.黑曲霉生长前后MEA的光谱分析。(a) MEA生长前(黑色)和生长后(红色)的紫外/可见吸光度光谱,以及两种光谱(蓝色)的差异。(b) 不同浓度柠檬酸在水中的紫外/可见吸光度光谱。(c)黑曲霉生长前(黑色)和后(红色)采样MAE的FT-IR光谱。1174、1365、1750 cm −1 处的峰显示了柠檬酸的典型峰。(为了解释此图例中对颜色的引用,读者可以参考本文的网络版本。

The species-selectivity of MEA-hybridized CNT-FET was further tested by observing the signal change under the growth of a yeast species, S. cerevisiae. It is reported that S. cerevisiae has a lag phase of about 6 h, followed by a log phase of ∼6 h, after which it reaches the stationary phase [40,41]. For this experiment, 20 μL of 1 mg/mL S. cerevisiae solution was dropped on the MEA just above the CNT channel. As shown in Fig. 4a, the current showed no significant change up to 6 h, which corresponds to the lag phase. Afterwards, the yeast colony showed exponential growth (log phase) and reached the stationary phase at 16 h, as observed with optical microscopy. The change in the CNT-FET current signal was also in accordance with this phase change. In case of pH, yeast is reported to generate ethanol as main metabolite, and therefore the pH is expected to increase [42]. Indeed, Fig. 4b shows that the pH increased after 6 h at the beginning of the log phase. Meanwhile, the effect of ethanol to the CNT-FET is known to increase its current [43]. In the case of other microbial organisms, we attempted to monitor activity of Penicillium polonicum (P. polonicum) (Fig. S10) [44]. However, there was little change in the CNT-FET conductivity. This shows that the CNT-FET can be selective towards specific fungal species.
通过观察酵母菌 S. cerevisiae 生长下的信号变化,进一步测试了 MEA 杂交 CNT-FET 的物种选择性。据报道,酿酒酵母的滞后期约为6 h,随后是∼6 h的对数期,之后达到固定期[40,41]。对于该实验,将 20 μL 的 1 mg/mL 酿酒酵母溶液滴在 CNT 通道正上方的 MEA 上。如图4a所示,电流在6 h内没有显著变化,这与滞后阶段相对应。之后,酵母菌落呈指数增长(对数阶段),并在16小时达到固定期,如光学显微镜观察的那样。CNT-FET电流信号的变化也与这种相位变化相一致。据报道,在pH值的情况下,酵母产生乙醇作为主要代谢物,因此pH值预计会增加[42]。事实上,图4b显示,在对数阶段开始时,pH值在6小时后增加。同时,已知乙醇对CNT-FET的影响会增加其电流[43]。对于其他微生物,我们试图监测青霉(P. polonicum)的活性(图S10)[44]。然而,CNT-FET电导率变化不大。这表明CNT-FET可以对特定的真菌物种进行选择。

Fig. 4
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Fig. 4. Microbial activity monitoring for yeast and competitive growth against A. niger. (a) The CNT-FET current and (b) MEA pH change in time for yeast Saccharomyces cerevisiae (S. cerevisiae). (c) The CNT-FET current change and (d) MEA pH change in time for simultaneous growth of fungus A. niger and yeast S. cerevisiae. The inset in (a) shows a colony of S. cerevisiae (white) grown above MEA-hybridized CNT-FET after 24 h (scale bar: 3 mm). The inset in (c) shows one colony (white, bottom) of S. cerevisiae and another colony (black, top) of A. niger after 72 h growth (scale bar: 3 mm). The dashed lines in the inset in (a) and (c) serve as optical guidances that show the positions of the samples and electrodes.
图 4.微生物活性监测,监测酵母和针对黑曲霉的竞争性生长。(a) 酵母酿酒酵母 (S. cerevisiae) 的 CNT-FET 电流和 (b) MEA pH 值随时间的变化。(c) CNT-FET电流变化和(d)MEA pH值随时间变化,促进真菌黑曲霉和酿酒酵母的生长。(a)中的插图显示了24小时后在MEA杂交的CNT-FET上方生长的酿酒酵母菌落(白色)(比例尺:3 mm)。(c)中的插图显示了生长72小时后酿酒酵母的一个菌落(白色,底部)和另一个黑曲霉菌落(黑色,顶部)(比例尺:3 mm)。(a)和(c)中插图中的虚线用作光学引导,显示样品和电极的位置。

To investigate the utility of our system in the simultaneous monitoring of the microbial activity of several species, we grew two different species (yeast and fungus) and observed the superposition effect from the two species. The two species (S. cerevisiae and A. niger) were applied on the MEA-hybridized CNT-FET simultaneously and were incubated together (Supporting Movie 2). The final signal showed a superposed response from the individual species (Fig. 4c). The signal showed a clear signal increase at ∼6 h, where the start of log-phase of yeast S. cerevisiae is expected. After ∼24 h, another signal increase was observed where the start of log-phase of A. niger is expected. Similarly, pH signal increase at the start of log-phase of yeast S. cerevisiae (∼6 h) and A. niger (∼24 h) (Fig. 4d). This shows that our platform can be used to simultaneously monitor signals of various species in real time.
为了研究我们的系统在同时监测几个物种的微生物活性中的效用,我们培养了两个不同的物种(酵母和真菌),并观察了两个物种的叠加效应。将两个物种(酿酒酵母和黑曲霉)同时应用于MEA杂交的CNT-FET上,并一起孵育(支持视频2)。最终信号显示单个物种的叠加响应(图4c)。信号在∼6小时时显示出明显的信号增加,预计酵母酿酒酵母的对数期开始。∼24 小时后,观察到另一个信号增加,预计黑曲霉的对数期开始。同样,酵母酿酒酵母(∼6小时)和黑曲霉(∼24小时)的对数期开始时pH信号增加(图4d)。这表明我们的平台可用于同时实时监测各种物种的信号。

As a control experiment, the direct effects of citric acid and ethanol on the CNT-FET signal were observed (Fig. 5). In case of citric acid, a major by-product of A. niger, CNT-FET current increase (Fig. 5a) and pH decrease (Fig. 5b) were observed. Therefore, in case of Aspergillus species, the CNT-FET current change can be attributed to the change in the major by-product of the fungal metabolism, which is citric acid. Likewise, ethanol, a major by-product of S. cerevisiae, appears to affect the CNT-FET current (Fig. 5c) and pH increase (Fig. 5d). The above shows that the major by-product of the fungal metabolism of each species can be attributed to the change of pH and CNT-FET current. Since the amount of ethanol generated by yeast is benign to the yeast growth conditions, this control experiment is appropriate for elucidating the effect of ethanol on CNT-FET signal change. There is the possibility that yeast can generate other metabolites such as maltose or glucose [45]. However, sugars show insignificant influence on the conductivity of the CNT-FET. During a long period of time, the composition of the MEA media can vary considerably by metabolite change and accumulation. According to our control experiments, some metabolites (citric acid, for example) have bigger effect on the conductivity of CNT-FETs compared to others.

Fig. 5
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Fig. 5. CNT-FET response and pH change to citric acid and ethanol. (a) Subsequent response of CNT-FET to 0.1 M citric acid and (c) 0.1 M ethanol in deionized water. (b) Subsequent pH change to 0.1 M citric acid and (d) 0.1 M ethanol in deionized water.
图 5.CNT-FET响应和pH值对柠檬酸和乙醇的变化。(a) CNT-FET在去离子水中对0.1 M柠檬酸和(c)0.1 M乙醇的后续响应。(b) 随后在去离子水中将 pH 值变为 0.1 M 柠檬酸和 (d) 0.1 M 乙醇。

4. Conclusions 4. 结论

An effective system for monitoring microbial activity should be able to track the different stages in their lag, log, and stationary phases during their growth. In this case, a more plausible approach would be to monitor the effect of total metabolites instead of a few selected one. For this purpose, we demonstrated a real- time detection system using MEA-hybridized CNT-FET for microbial activity monitoring. The signal change was based on the conductance change of the CNTs due to the metabolites during the growth of fungi and yeast. The CNT-FET signals represented more closely the microbial growth phases compared to pH changes. Unlike conventional biosensors, this platform enables us to investigate the physicochemical change of the medium caused by the microbial growth and the metabolites of the microorganisms. Further work would involve verification of the effect of the individual metabolites using techniques such as mass spectroscopy in conjunction. Since this strategy can monitor microbial activity in real time, we expect that the platform can be used as a supplement or an alternative to current time-consuming, costly and difficult microbial monitoring procedures and improve microbial monitoring performance.

Acknowledgments 确认

This research was supported by the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT and Future Planning (2015R1A2A2A04002733, 2016M3A7B4909581).

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References 引用

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Minjun Park received his M.S. degree in the School of Mechanical Engineering of Korea University, Korea. His current research topic includes biomimetic material assembly and electromechanical characterization of nanoscale biomaterials using scanning probe microscopy.

Hyun Soo Kim received B.S. degree in the School of Mechanical Engineering of Korea University, Korea. His current research topic includes the design and synthesis of functional materials for nanodevices for sensors and actuators.

Taewan Kim received B.S. degree in Department of Physics from Myongji University, Korea. He is currently a M.S. graduate student in Mechanical Engineering, Korea University, Seoul, Korea. His current research topic is nanoelectronic detection in biomedical drug screening using nanoscale based platform.

Junhyup Kim received his M.S. degree in the School of Mechanical Engineering of Korea University, Korea. His current research interests are focused in the development of carbon nanotube-based biosensors for the detection of pathogens and microorganisms.

Sungchul Seo is currently a professor at the Department of Industrial Health, Catholic University of Pusan, Busan, Korea. His current research interests include industrial safety management, organic chemistry, hazardous material risk assessment and indoor air pollution.

Byung Yang Lee is currently a professor at Department of Mechanical Engineering, Korea University, Seoul, Korea. His current research interests include biochemical nanosensors, nanomaterial synthesis, and soft actuators.

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